The present disclosure relates generally to methods for preparing biomaterial arrays and methods for using the biomaterial arrays. More particularly, the present disclosure relates to hydrogel arrays, methods for preparing hydrogel arrays and methods for screening cell-substrate interactions using the hydrogel arrays. The hydrogel arrays include individual hydrogel posts that are completely isolatable, allowing for systematic and independent control of the chemical composition and physical dimensions of each hydrogel post.
The development of most tissue types involves a complex interplay of multiple signals leading to controlled precursor cell differentiation into mature, tissue-specific cell types. For example, mesenchymal stem cells (MSCs) may be differentiated in vitro into osteoblasts, chondrocytes, myoblasts, adipocytes, neurons, and endothelial cells by exposure to a variety of growth factors. Exposure to growth factors may be controlled by the media and the substrates upon which the cells are cultured. Substantial progress has been made in the development of defined media, but only more recently has the role of substrates and cell-substrate adhesion on cell growth been examined.
Based on studies to determine defined media, it has become apparent that the substrate is important for successful cellular growth and tissue generation. For example, it has been demonstrated that attachment to the substrate by human embryonic stem cells may contribute to the variability in whether the cells remain undifferentiated or undergo differentiation. Therefore, it is important to not only identify cell culture media for successful cell culture conditions, but to also identify defined substrates.
Screening well-defined surfaces in an array format allows rapid identification of specific molecules that promote cellular adhesion, cellular spreading, proliferation, migration and differentiation, as well as molecules that regulate cell behavior. Biomaterial arrays such as self-assembled monolayers (“SAMs”) in array formats (i.e., SAM arrays) have been constructed that present ligands to cells plated onto the array. A SAM is an organized layer of amphiphilic molecules in which one end of the molecule exhibits a specific, reversible affinity for a substrate and the other end of the molecule has a functional group. The use of alkanethiols to construct SAM arrays allow for the formation of reproducible SAM arrays and surfaces. SAM arrays may be used to identify specific ligands or epitopes that promote cellular attachment, spreading, proliferation, migration and differentiation. Additionally, SAM arrays may be patterned such that ligands will be presented to the cells in defined areas of the array.
Biomaterial array patterning approaches have been developed to spatially localize ligands to create spatially and chemically-defined cell culture substrates. Microcontact printing, for example, generates patterned SAM arrays by “inking” alkanethiolate molecules onto a flexible elastomeric stamp and stamping the alkanethiolates onto a gold surface, which transfers a pattern of ligands onto the gold substrate. The remaining areas of bare gold are then “backfilled” with a second alkanethiolate species to generate a bio-inert SAM surrounding the stamped hydrophobic alkanethiolate domains. The substrates are then bathed in a solution of ligands that spontaneously adsorb to the hydrophobic alkanethiolate regions to create patterned islands for cell attachment. Microfluidics approaches for SAM array patterning typically use elastomeric stamps with microscale features that form channels when passively adhered to a SAM. Localized ligand conjugation can then be achieved by flowing reaction solutions through the channels exposing them to reactive terminal moieties presented by the underlying SAM. Photochemistry in combination with micro-patterned photomasks can be used to create patterned SAM arrays by selectively protecting a reactive terminal moiety and then selectively deprotecting the terminal moiety to locally immobilize ligands on the SAM. SAM array patterning can also be accomplished by locally destroying/removing regions of a fully formed SAM, then reforming new SAMs in the destroyed regions.
While biomaterial arrays such as SAM arrays provide an excellent model substrate for investigating the effects of an immobilized ligand on cell behavior, preparing SAM array platforms using less labor intensive processes are needed to make SAM array use more widespread. Accordingly, there exists a need for alternative methods for preparing biomaterial arrays to identify surfaces that will support survival and growth of cells in culture, allow rapid identification of specific molecules that promote cellular adhesion, cellular spreading, proliferation, migration, differentiation and regulate cellular behavior.